Combinatorial optimization of cyanobacterial 2,3-butanediol production

A vital goal of renewable technology is the capture and re-energizing of exhausted CO₂ into usable carbon products. Cyanobacteria fix CO₂ more efficiently than plants, and can be engineered to produce carbon feedstocks useful for making plastics, solvents, and medicines. However, fitness of this technology in the economy is threatened by low yields in engineered strains. Robust engineering of photosynthetic microorganisms is lagging behind model microorganisms that rely on energetic carbon, such as Escherichia coli, due in part to slower growth rates and increased metabolic complexity. In this work we show that protein expression from characterized parts is unpredictable in Synechococcus elongatus sp. strain PCC 7942, and may contribute to slow development. To overcome this, we apply a combinatorial approach and show that modulation of the 5'-untranslated region (UTR) can produce a range of protein expression sufficient to optimize chemical feedstock production from CO₂.     

The primary structure of Pseudomonas cytochrome c peroxidase

The primary structure of Pseudomonas cytochrome c peroxidase is presented. The intact protein was fragmented with cyanogen bromide into five fragments; partial cleavage was observed at a Met-His bond of the protein. The primary structure was established partly by automatic Edman degradations, partly by manual sequencing of peptides obtained with trypsin, thermolysin, chymotrypsin, pepsin, subtilisin and Staphylococcus aureus V8 endopeptidase. The order of the cyanogen bromide fragments was further confirmed by overlapping peptides obtained by specific cleavage of the whole protein. Pseudomonas cytochrome c peroxidase consists of 302 amino acid residues giving a calculated M_r of 33 690.     

Strategies for rapid assessment of fungal diversity

The problems of estimating fungal diversity are considerable, due especially to the large numbers of species even in small study sites, only a small proportion of which are adequately characterized, and the lack of trained staff and appropriate identification manuals. These difficulties are multiplied in the tropics. Strategies for providing preliminary assessments of fungal diversity which can be carried out within short time-frames are discussed, which will allow initial site valuations to be made for conservation purposes. The necessity of identifying all species discovered is considered, and the need emphasized for strictly defined sampling protocols which allow proper comparison between sites. The advantages and disadvantages of preliminary diversity analyses involving different taxonomic and ecological groups, fungus/plant associations, spatial sampling, soil and litter analysis, molecular methods and indirect assessment are considered.     

Architecture and function of metallopeptidase catalytic domains

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single‐step reaction involving a solvent molecule, a general base/acid, and a mono‐ or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal‐binding motif (HEXXH), which includes two metal‐binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ～130–270‐residue catalytic domains, which are usually preceded by N‐terminal pro‐segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C‐terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N‐terminal subdomain spanning a five‐stranded β‐sheet, a backing helix, and an active‐site helix. The latter contains most of the metal‐binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C‐terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met‐turn—and a C‐terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.     

Landscape structure influences tree density patterns in fragmented woodlands in semi‐arid eastern Australia

Landscape and local‐scale influences are important drivers of plant community structure. However, their relative contribution and the degree to which they interact remain unclear. We quantified the extent to which landscape structure, within‐patch habitat and their confounding effects determine post‐clearing tree densities and composition in agricultural landscapes in eastern subtropical Australia. Landscape structure (incorporating habitat fragmentation and loss) and within‐patch (site) features were quantified for 60 remnant patches of Eucalyptus populnea (Myrtaceae) woodland. Tree density and species for three ecological maturity classes (regeneration, early maturity, late maturity) and local site features were assessed in one 100 × 10 m plot per patch. All but one landscape characteristic was determined within a 1.3‐km radius of plots; Euclidean nearest neighbour distance was measured inside a 5‐km radius. Variation in tree density and composition for each maturity class was partitioned into independent landscape, independent site and joint effects of landscape and site features using redundancy analysis. Independent site effects explained more variation in regeneration density and composition than pure landscape effects; significant predictors were the proportion of early and late maturity trees at a site, rainfall and the associated interaction. Conversely, landscape structure explained greater variation in early and late maturity tree density and composition than site predictors. Area of remnant native vegetation within a landscape and patch characteristics (area, shape, edge contrast) were significant predictors of early maturity tree density. However, 31% of the explained variation in early mature tree differences represented confounding influences of landscape and local variables. We suggest that within‐patch characteristics are important in influencing semi‐arid woodland tree regeneration. However, independent and confounding effects of landscape structure resulting from previous vegetation clearing may have exerted a greater historical influence on older cohorts and should be accounted for when examining woodland dynamics across a broader range of environments.     

Correlation between the compact regions predicted by the Average Distance Map (ADM) and the biologically active sites in atrial natriuretic peptide

The prediction of the short-range compact regions of human atrial natriuretic peptide (a-hANP), one of the biologically active peptides, has been made by means of the Average Distance Map(ADM). We found out that the location of the predicted short-range compact regions is consistent with the structural units determined by the NMR analysis (Kobayashiet al., 1988). Furthermore, the short-range compact regions correspond well to the biologically active areas of atriopeptin (103–125)-amide (which is homologous peptide toa-hANP), detected by the glycine substitution technique (Konishiet al., 1987). The results suggest that a predicted short-range compact region can be regarded as a possible active site in a biologically active peptide.     

Observation and quantitation of metal-binding sites in the sex steroid-binding protein of human and rabbit sera using the luminescent probe terbium

The first direct evidence for specific metal-binding sites in pure human and pure rabbit sex steroid-binding protein (SBP) is obtained using the luminescent lanthanide terbium. Terbium, a probe for calcium sites in proteins, provided protection of the SBP steroid-binding activity in diluted human serum samples equivalent to that provided by calcium. Pure SBP, first treated with ethylenediaminetetraacetate, was dialyzed against buffer containing TbCl₃. After gel filtration to remove nonspecifically bound terbium, the protein was denatured in urea. The amount of protein-bound terbium was determined by luminescence enhancement of the lanthanide using the chelator dipicolinate, yielding four metal-binding sites per mole of dimer protein from both species.     

一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 *

羟基化氨基酸是一种新型氨基酸衍生物,可广泛用作化工材料的前体物及医药合成的中间体。将来源于Nostoc minutum的新型L-亮氨酸5-羟化酶 (NmLEH) 通过重组质粒在大肠杆菌中异源表达。结果表明,在BL21(DE3) 宿主细胞中,诱导温度为25℃,IPTG诱导浓度为0.5mmol/L,诱导10h时,蛋白质表达量最高 (0.45mg/ml);通过Ni-亲和层析和凝胶过滤层析两步分离纯化获得了高度纯化的重组NmLEH蛋白;对NmLEH的酶学性质进行了表征,该酶的最适反应温度为25℃,最适pH 为7.5,在pH 7.0~9.0较为稳定,最适底物为亮氨酸和甲硫氨酸;同源序列分析表明NmLEH属于亚铁和α-酮戊二酸依赖性双加氧酶家族[Fe(II)/αKG-Dos],并预测了该酶的保守催化活性位点(H150、D152、H236);通过同源建模得到了该蛋白质的模拟结构,分析了该蛋白质催化活性中心的形成机制。